Due to their high biocompatibility, high water content and excellent permeability to nutrients and metabolites, hydrogels have been extensively studied as biomaterials to be used in various biomedical applications, such as implants, drugs and cell delivery carriers. Hydrogels may be prepared from naturally occurring or synthetic polymers and be formed in a chemical and physical three-dimensional crosslinked network. Over the last decade, trends in hydrogel study have shifted to in situ-forming hydrogels, which are formed at the actual site following the in vivo injection of a polymer solution.
In situ forming hydrogels may be used in injectable hydrogel systems. Based on minimally invasive techniques, injectable hydrogel systems have attracted intensive attention because they make up easy formulations which are comfortable to patients. These systems may composed of injectable fluids which can form hydrogels in vivo, e.g., in tissues, organs, or coeloms before being solidified upon injection using minimally invasive methods.
For example, injectable hydrogel systems do not require surgical procedures for their implantation, but various drugs and bioactive molecules can be encapsulated easily into the hydrogel by simple mixing. It is possible to fill defected or depressed sites of the body cavities with injectable hydrogel. Injectable hydrogel systems have poor mechanical properties, but enjoy the advantages of exhibiting high cell seeding efficiency, being useful as carriers for bioactive drugs such as peptides, proteins and DNA, and effectively delivering nutrients to cells.
In-situ hydrogel formation may be the result of chemical crosslinking achieved by UV-polymerization or Michael addition or by physiochemical crosslinking such as ionic bonding, stereocomplex formation or thermosensitive binding following hydrophobic interaction. When formed by chemical crosslinking, the in-situ formed hydrogels are restricted in use due to their cytotoxicity and poor in vivo safety which results mainly from the use of toxic additives such as a photoinitiator or a crosslinking agent. In contrast, the in-situ forming hydrogels prepared by physiochemical crosslinking are in principle free of the toxic additives, but suffer from the disadvantages of low mechanical strength and stability.
It is particularly difficult in the case of photosensitive hydrogels to uniformly mix a solution of the precursor polymer solution with cells or drugs due to the high viscosity thereof, and these are also disadvantageous in that their preparation takes a long period of time. On the other hand, when the stereocomplexed hydrogel degrades, it produces acidic by-products which cause cytotoxicity and necrosis to the surrounding tissue.
In order to overcome these problems, an enzyme-triggered in-situ forming hydrogel has recently been developed. This crosslinked hydrogel results from the in situ polymerization of a polymer in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). In addition to having excellent in situ safety, the enzyme-triggered in-situ forming hydrogel enjoys the advantage of the chemically crosslinked hydrogel, that is, high mechanical strength.
The enzyme-triggered in-situ forming hydrogels developed so far are as follows: dextran-tyramine (dec-TA) (Rong Jin et al, Biomaterials 2007), hyaluronic acid-tyramine (HA-TA) (Motoichi Kurisawa et al, Chem. Commun. 2005), gelatin-hydroxypropionic acid (GHPA) (Lishan Wang et al, Biomaterials 2009), gelatin-tyramine(GTA) (Shinji Sakai et al, Biomateirals 2009), and alginic acid-hydroxyphenylacetic acid (AHPA) (Shinji Sakai et al, Acta Biomaterialia 2007).
Hydrogels which are formed in situ by enzyme-mediated crosslinking from hyaluronic acid-tyramine are commercially available from LifeCore and there have been many related PCT and U.S. patents issued. The HRP-mediated coupling reaction of phenol moieties in the polymer backbones occurs via a carbon-carbon bond at the ortho positions and/or via a carbon-oxygen bond between the carbon atom at the ortho position and the phenoxy oxygen.
The physiochemical properties of the formed hydrogel, such as gelation time, mechanical strength, biodegradability, etc., can be easily controlled with the concentration ratio of HRP to H2O2. However, these enzyme-triggered hydrogels suffer from the disadvantages of having poor stability and mechanical properties, attributable to the solubility of the polymer solution and the reactivity of phenol-phenol coupling.
For example, the amount of the polymer solutions in these hydrogels is restricted to the range of from as low as 1 to 5 wt % due to the high viscosity thereof. In practice, the high viscosity makes it difficult to uniformly mix cells or drugs within the hydrogels.
The low solubility of gelatin-tyramine (GTA) may lead to an opaque hydrogel and permit the polymer solution to be at a concentration of 5 wt % or less. Because the rate of solubility of gelatin is affected by other factors (especially low temperatures) there is a need to solve this problem.
As for gelatin-hydroxypropionic acid (GHPA) hydrogels, their mechanical strength is 600 Pa at best. This poor mechanical strength is attributed to the fact that the phenol moieties of the polymer backbone are directly bonded to each other. The mobility of the phenol moieties plays an important role in forming a phenol-phenol bond because the formation of radicals for phenol-phenol bonds requires that the distance between HRP molecules and phenol moieties be at least several angstroms (Å). Thus, drawbacks occur in structures where the phenol moieties are directly bonded.
Therefore, there is a need for an in situ forming hydrogel that is superior in terms of biostability and mechanical strength.